Orthogonal Peptide-Templated Labeling Elucidates Lateral ETAR/ETBR Proximity and Reveals Altered Downstream Signaling

Fine-tuning of G protein-coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one-step labeling method of multiple membrane-embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this approach to four receptors of the cardiovascular system: the endothelin receptor A and B (ET_AR and ET_BR), angiotensin II receptor type 1, and apelin. Wild type-like G protein activation after N-terminal modification was demonstrated for all receptor species. Using FRET-competent dyes, a constitutive proximity between hetero-receptors was limited to ET_AR/ET_BR. Further, we demonstrate, that ET_AR expression regulates the signaling of co-expressed ET_BR. Our orthogonal peptide-templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling.     

Biosynthetic Plasticity Enables Production of Fluorinated Aurachins

Enzyme promiscuity has important implications in the field of biocatalysis. In some cases, structural analogues of simple metabolic building blocks can be processed through entire pathways to give natural product derivatives that are not readily accessible by chemical means. In this study, we explored the plasticity of the aurachin biosynthesis pathway with regard to using fluoro- and chloroanthranilic acids, which are not abundant in the bacterial producers of these quinolone antibiotics. The incorporation rates of the tested precursor molecules disclosed a regiopreference for halogen substitution as well as steric limitations of enzymatic substrate tolerance. Three previously undescribed fluorinated aurachin derivatives were produced in preparative amounts by fermentation and structurally characterized. Furthermore, their antibacterial activities were evaluated in comparison to their natural congener aurachin D.     

Zafirlukast Induces VHL- and HIF-2α-Dependent Oxidative Cell Death in 786-O Clear Cell Renal Carcinoma Cells

Mutations in the Von Hippel–Lindau (VHL) gene are the driving force in many forms of clear cell renal cell carcinoma (ccRCC) and promote hypoxia-inducible factor (HIF)-dependent tumor proliferation, metastasis and angiogenesis. Despite the progress that has already been made, ccRCC generally remain resistant to conventional therapies and ccRCC patients suffer from metastasis and acquired resistance, highlighting the need for novel therapeutic options. Cysteinyl leukotriene receptor 1 (CysLTR1) antagonists, like zafirlukast, are administered in bronchial asthma to control eicosanoid signaling. Intriguingly, long-term use of zafirlukast decreases cancer risk and leukotriene receptor antagonists inhibit tumor growth, but the mechanisms still remain unexplored. Therefore, we aim to understand the mechanisms of zafirlukast-mediated cell death in ccRCC cells. We show that zafirlukast induces VHL-dependent and TNFα-independent non-apoptotic and non-necroptotic cell death in ccRCC cells. Cell death triggered by zafirlukast could be rescued with antioxidants and the PARP-1 inhibitor Olaparib, and additionally relies on HIF-2α. Finally, MG-132-mediated proteasome inhibition sensitized VHL wild-type cells to zafirlukast-induced cell death and inhibition of HIF-2α rescued zafirlukast- and MG-132-triggered cell death. Together, these results highlight the importance of VHL, HIF and proteasomal degradation in zafirlukast-induced oxidative cell death with potentially novel therapeutic implications for ccRCC.     

Detection of cauliflower mosaic virus by the polymerase chain reaction: testing of food components for false-positive 35S-promoter screening results

 Today DNA-based techniques are very common for the detection of genetically modified organisms (GMOs) in food products. For fast and easy detection of GMOs, polymerase chain reaction (PCR) screening methods, which amplify common transgenic elements, are applied in routine analysis. These techniques do not allow differentiation between GMOs and the natural occurrence of transgenic elements, such as the 35S-promoter of cauliflower mosaic virus (CaMV) or the NOS-terminator of Agrobacterium tumefaciens, and thus may result in false-positive detection of GMOs. In this study we evaluated three different existing 35S screening systems and report the development of two new CaMV-specific PCR systems. These PCR systems based on CaMV-specific genes allow the identification of positively screened 35S food samples as naturally virus-infected products or plants. Seven food samples tested positive in routine 35S screening analysis and negative in GMO specific systems were investigated using the new virus-specific PCR systems. In all seven samples CaMV was detected. Received: 26 April 1999 / Revised version: 28 June 1999     

Peanut varieties with reduced Ara h 1 content indicating no reduced allergenicity

Peanut allergy is a major cause of food‐induced severe anaphylactic reactions. To date, no medical care is available to prevent and treat peanut allergy and therefore hypoallergenic peanut varieties are of considerable health political and economic interest. Major allergens that induce IgE‐responses in peanut‐sensitive patients are Ara h 1, Ara h 2 and Ara h 3/4. In order to identify hypoallergenic peanuts, commercially locally available peanut varieties were screened for their allergen content. Ara h 1‐deficient peanuts from Southeast Asia were identified by SDS‐PAGE, immunoblotting, inhibition assays and ELISA. 2‐D PAGE analyses demonstrated the different compositions of the tested extracts and revealed a number of variations of the allergen patterns of peanuts from different varieties. Mediator release experiments of these peanut extracts demonstrated similar allergenicities as compared with standard peanut extract. These results indicate that the allergenicity of peanuts with reduced Ara h 1 content might be compensated by the other allergens, and thus do not necessarily cause a reduction of allergenicity.